Human tissue-type plasminogen activator (t-PA), a single chain serine protease of Mr 68,000, is a key physiological regulator of fibrinolysis. It converts the zymogen plasminogen into plasmin, the enzyme which degrades the fibrin network of the thrombus (Collen (1980) Thromb. Haemostasis 43:77-82; Rijken and Collen (1981) J. Biol. Chem. 256:7035-7041). Apparently, in the presence of a clot, both t-PA and plasminogen bind to fibrin and form a ternary complex in which plasminogen is efficiently activated (Holyaerts et al. J. Biol. Chem. 257:2912-2929; Ranby (1982) Biochim. Biophys. Acta 704:461-469). The affinity of t-PA for fibrin makes t-PA a clot-specific and useful thrombolytic agent (Van de Werf et al. (1984) Circulation 69:605-610), which has been approved for human use in the treatment of acute myocardial infarction and is expected to be approved for the treatment of other thrombotic disorders including pulmonary embolism and deep vein thrombosis. T-PA produces limited conversion of plasminogen in the absence of fibrin, so its effects tend to be localized at the site of a thrombus with limited systemic proteolysis. In addition to binding to plasminogen and fibrin, t-PA binds to a fast-acting plasminogen activator inhibitor (PAI-1), which has been identified in blood plasma and in the culture medium of various cells, and which regulates t-PA activity by complexing with and neutralizing the serine protease (Van Mourik et al. (1984) J. Biol. Chem. 259:14914-14921; Colucci et al. (1985) J. Clin. Invest. 75:818-814; Almer and Ohlin (1987) Thromb. Research 47:335-339). PAI-1 is also referred to as PAI in this application.
The turnover of t-PA in plasma is rapid with an in vivo functional half-life of 2 to 6 min, depending on the species (Korninger et al. (1981) Thromb. Haemostasis 46:658-661; Verstraete et al. (1985) J. Pharmacol. Exp. Ther. 235:506-512). Exogenously introduced t-PA is rapidly taken up and accumulated in the liver (Fuchs et al. (1985) Blood 65:539-544; Emeis et al. (1985) Thromb. Haemostasis 54:661-664). Such observations suggest that clearance of plasma t-PA, like that for certain other serum glycoproteins (Ashwell and Harford (1982) Ann. Rev. Biochem. 51:531-554) may be mediated through an interaction with a specific hepatic cell surface receptor, followed by internalization and degradation within the cell. The recent demonstration of a novel high affinity uptake system for recombinant t-PA on rat hepatocytes supports this hypothesis (Bakhit et al. (1987) J. Biol. Chem. 262:8716-8720).
There is a significant need for a modified t-PA which retains its thrombolytic effectiveness in vivo, but which is not subject to rapid degradation following its administration. Such a modified t-PA would have enhanced utility for treatment of thrombotic disorders, such as acute myocardial infraction.